Immunocytochemistry/immunohistochemistry and Western blot

Human tumour cell lines harbouring wildtype or mutated Ras [32, 33], primary tumour tissues and the corresponding normal tissues were employed to detect the immunoreactivities of the scFv antibodies by immunohistochemical staining. The soluble scFv antibodies were used as the primary antibody, whereas Anti-E-tag monoclonal antibody conjugated with HRP served as the second antibody to bind E-tag protein ligated to the end of the scFv antibody. The scFv immunoreactivity with p21Ras was further detected by Western blot in the tumour cell lines MDA-MB-435, MDA-MB-231 and SKOV3 (ATCC USA) and the normal cell line KMB17 (normal human embryonic diploid lung fibroblast cell, constructed by the Institute of Medical Biology, Chinese Academy of Medical Sciences). β-actin was used as a control, with anti-β-actin monoclonal antibody conjugated with HRP.